Antibiotic resistance is growing at a rapid pace and is a major threat to human health. The need for new antibiotics is eminent, though there is a lack of research programs in the industry devoted to this cause. At the Institute for Genomic Biology at the University of Illinois, we aim to identify and study potential antibiotics by mining microbial genomes. Phosphonates and phosphinates, compounds containing one or two direct carbon-phosphate (C-P) bonds, respectively, are a growing class of natural products with antibacterial and antifungal activity. The biosynthetic pathways that produce these compounds have remained largely unexplored until recently. The study of antibiotic biosynthetic pathways is necessary for our goal to eventually produce analogs through engineering of the biosynthetic machinery. Elucidation of the activity of specific enzymes in these pathways will help better predict the structure of new, undescribed C-P containing natural products and the function of the biosynthetic machinery that produces them. Phosphoenol pyruvate mutase (Ppm) is responsible for constructing the first C-P bond in the biosynthesis of the antifungal agent phosphinothricin tripeptide (PTT). In close proximity to the gene for this enzyme is a gene encoding for hydroxyethyl phosphonate dioxygenase (HEPD), which converts 2-hydroxyethylphosphonate (2-HEP) to hydroxy-methylphosphonate (HMP), a later step in PTT biosynthesis. Two enzymes have been identified in Nitrosopumilus maritimus, an archaeal marine species, one of which is homologous to Ppm and the other is a potential downstream dioxygenase with homology to HEPD. These enzymes represent the first examples of archaeal involvement in phosphonate biosynthesis and provide a link to methane production in the oceans, a major contributor to the greenhouse effect. We have already shown the new dioxygenase to convert 2-HEP to methylphosphonate (MPn). We believe that this HEPD homolog, or methylphosphonate synthase (MpnS), catalyzes this transformation via a similar hydroperoxylation mechanism predicted for HEPD. This proposal involves comparison of native activity of both dioxygenases by analysis of kinetic parameters and identification of reaction intermediates. Next, the substrate flexibility of MpnS will be studied to aid in elucidating its mechanism. Finally, the active sites of both enzymes will be probed through mutagenesis. The work proposed will expand on current research of the mechanism of HEPD, will aid in further elucidating the function of MpnS in N. maritimus, and will provide a better understanding of both mechanisms so as to afford better predictions a priori of in vivo function of new HEPD homologs for use in antibiotic bioengineering. PUBLIC HEALTH RELEVANCE: Antibiotic resistance is a major threat to human health and it is imperative that new classes of antibiotics are developed. This work will investigate how certain types of compounds with antibiotic properties are made by microbial organisms and will enable the identification of additional antibiotic candidates produced by other organisms based on their genomes.